Pentoxifylline Ameliorates Cardiac Fibrosis, Pathological Hypertrophy, And Cardiac Dysfunction In Angiotensin Ⅱ-induced Hypertensive Rats

BackgroundInflammation is a common pathophysiological characteristic in many types of cardiovascular diseases including coronary arterial disease, heart failure, and hypertension. Activation of the renin-angiotensin system (RAS) is thought to be a major mechanism underlying inflammation in hypertension.Angiotensin (ANG) Ⅱ, a key mediator of the RAS, induces the synthesis and release of proinflammatory cytokines such as tumor necrosis factor (TNF)-a and interleukin (IL)-1β through activation of several intracellular signaling pathways including nuclear factor-kappa B (NFkB).A growing body of evidence has suggested that myocardial inflammation plays a key role in regulation of cardiac remodeling including myocardial fibrosis and pathological hypertrophy. Increased myocardial fibrosis and pathological hypertrophy may lead to augmented ventricular wall stiffness and impaired cardiac diastolic function, and therefore represent an important risk factor for heart failure in both experimental models and patients. Interventions that reduce the levels of proinflammatory cytokines may significantly ameliorate these effects and prevent heart failure.Pentoxifylline (PTX) is a methylxanthine derivative and nonspecific phosphodiesterase inhibitor that has been used to treat patients with occlusive peripheral vascular disorders for more than 30 years. In addition to its rheologic properties, PTX has anti-inflammatory, antiproliferative, and antifibrotic actions. Previous studies have suggested that systemic treatment with PTX suppresses the production of proinflammatory cytokines and produces beneficial effects in myocardial infarction, heart failure, and idiopathic dilated cardiomyopathy in both animals and humans. However, no study to date has examined the effects of PTX on myocardial damage and cardiac dysfunction in ANG Ⅱ dependent hypertension.ObjectiveIn this study, we tested the hypothesis that treatment with PTX would inhibit proinflammatory cytokines, prevent myocardial fibrosis and pathological hypertrophy, and ameliorate cardiac dysfunction in ANG Ⅱ induced hypertensive rats.Methods1.64 adult male Sprague-Dawley rats were randomly divided into 4 groups (n=16 for each group):(1) control alone (CON), (2) control+PTX (CON+PTX), (3) ANG Ⅱ alone (ANG Ⅱ), and (4) ANG Ⅱ+PTX.2. Hypertension was induced by chronic ANG Ⅱ infusion (200 ng/kg per minute) using osmotic minipumps (model 2002; Alzet) for 2 weeks as described previously. Rats that received saline alone served as control. Osmotic minipumps were implanted subcutaneously under ketamine-xylazine (100 and 10 mg/kg) anesthesia. PTX (30 mg/kg daily) was given in drinking water for 2 weeks, beginning at the same time as the ANG Ⅱ subcutaneous infusion.Average fluid consumption was established by a pliot study.Fluid intake and body weights were recorded daily at the same time.3. Blood pressure (BP) and heart rate were measured in some rats (n=8 for each group) by tail-cuff plethysmography (BP-98A; Softron Co, Tokyo, Japan) every other day. After 2 weeks of ANG Ⅱ infusion and PTX treatment,echocardiography was performed to assess cardiac remodeling and function. These animals were then killed by an intraperitoneal injection of an overdose of sodium pentobarbital (210 mg/kg) to collect blood samples and heart tissues for biochemical assay, molecular studies, and analysis of myocardial hydroxyproline.4. Other animals from each group were killed by an intraperitoneal injection of the same dose of sodium pentobarbital and transcardially perfused with 4% paraformaldehyde for histological examination.5. Statistical analysis:all data are expressed as mean ±SEM. Differences in mean values were analyzed using one-way or 2-way ANOVA followed by Newman-Keuls multiple-comparison post hoc test. P<0.05 was considered statistically significant.Results1. Pentoxifylline has no effect on angiotensin Ⅱ-induced hypertension1.1. There was no difference in the heart rate among the experimental groups (Fig. IB).1.2. ANGⅡ infusion for 2 weeks produced a rapid and progressive increase in mean BP when compared with CON rats (Fig.1 A).1.3. Notably, PTX treatment had no effect on mean BP in both ANG Ⅱ and CON rats.2. Pentoxifylline ameliorates cardiac hypertrophy and dysfunction in angiotensin Ⅱ-induced hypertensive rats (Table 2)2.1. Body weight was similar across the 4 groups at the end of the study period;2.2. ANG Ⅱ rats experienced an increase in heart weight to body weight ratio compared with CON rats;2.3. Echocardiographic studies revealed that ANG Ⅱ rats had greater RWT, decreased E/A ratio, markedly increased IVRT compared with ANG II+PTX rats;2.4. Ejection fraction was not significantly altered by ANG II infusion;2.5. PTX did not alter these parameters in CON rats.3. Pentoxifylline attenuates cardiac fibrosis in angiotensin Ⅱ-induced hypertensive rats3.1. Histological analysis showed that ANG Ⅱ rats had increased interstitial and perivascular myocardial fibrosis compared with CON rats (Figs.2A, B);3.2. There are higher myocardial hydroxyproline levels in ANG II rats compared with CON rats (Fig.2C);3.3. As shown in Figures 3A and C, both CON and CON+PTX rats had few a-SMA expressions in the heart;3.4. Compared with CON rats, ANG Ⅱ rats had markedly higher a-SMA expression in the heart;3.5. Compared with ANG Ⅱ rats, ANG Ⅱ+PTX rats had significantly less a-SMA expression in the heart.4. Pentoxifylline reduces myocardial pro- and anti-inflammatory cytokine expression in angiotensin Ⅱ-induced hypertensive rats4.1. Compared with CON rats, ANG Ⅱ rats showed increased mRNA and protein expressions of pro-and anti-inflammatory cytokines TNF-a, IL-1β,IL-6, and IL-10 in the heart (Fig.4);4.2. ANG +PTX rats showed reduced mRNA and protein expressions of TNF-a, IL-1β, IL-6, and IL-10 as compared with ANG Ⅱ rats;4.3. No differences in expression of pro- and anti-inflammatory cytokines were observed between CON+ PTX and CON rats;4.4. Immunohistochemical study revealed that macrophage infiltration in the perivascular space was markedly higher in ANG Ⅱ rats than CON rats (Figs.3B and D);4.5. Compared with ANG Ⅱ rats, ANG Ⅱ+PTX rats had less macrophage infiltration in the perivascular space.;4.6. CON+PTX rats had similar macrophage infiltration to CON rats.5. Pentoxifylline suppresses activation of myocardial NF-κB in angiotensin II-induced hypertensive rats5.1. ANG II has been shown to activate the nuclear factor NF-κB, which stimulates multiple proinflammatory cytokine expression;5.2. As shown in Figure 5, NF-κB p65 DNA binding activity and IkBa expression in the heart were similar between CON+PTX and CON rats;5.3. Compared with CON rats, ANG Ⅱ rats displayed markedly increased NF-κB p65 DNA binding activity and decreased IkBa protein expression;5.4. Compared with ANG Ⅱ rats, ANG Ⅱ+PTX rats showed reduced NF-κB p65 DNA binding activity and elevated IkBa protein expression.6. Pentoxifylline attenuates gene expression of genetic markers for cardiac remodeling in angiotensin II-induced hypertensive rats6.1. As shown in Figure 6, we found that ANGⅡ rats had significantly increased mRNA expression for collagen I, TGF-β,CTGF,β-MHC, MCP-1, ICAM-1, and VCAM-1 in the heart as compared with CON rats;6.2. ANG II-induced increase in these parameters were attenuated in ANG Ⅱ+PTX rats;6.3. PTX did not alter these parameters in CON rats.7. Pentoxifylline reduces circulating proinflammatory cytokine levels in angiotensin Ⅱ-induced hypertensive rats7.1. ANG Ⅱ rats had higher plasma levels of TNF-a and IL-1β than CON rats (Fig. 7).;7.2. ANG Ⅱ+PTX rats had lower plasma levels of TNF-a and IL-1β compared with ANG Ⅱ rats.ConclusionThe findings suggest that PTX ameliorates cardiac fibrosis, pathological hypertrophy, and cardiac dysfunction by suppressing inflammatory responses in angiotensin Ⅱ-induced hypertension, and that these benefits were independent of the blood pressure lowering effect. The PTX by its anti-inflammatory property may be a potential therapeutic option for the prevention of cardiac remodeling and dysfunction in ANG-II induced hypertension.